Commercial preparations of Bacillus thuringiensis are used worldwide for biological control of pest insects. The advantages of these bacterial insecticides are that they are highly selective for a very limited range of target insects and are biodegradable.
Commercial preparations of Bacillus thuringiensis can be applied right up to the time of harvest with no adverse effects.
Bacillus thuringiensis is a rodshaped, aerobic, spore forming bacterium uniquely characterized by the production during the sporulation process of one or more inclusions, referred to as parasporal crystals. These crystals are composed of high molecular weight proteins, referred to as delta-endotoxins. The delta-endotoxins are the active ingredient in available commercial preparations of Bacillus thuringiensis.
Many B. thuringiensis strains with different insect host spectra have been identified. They are classified into different subspecies based on their flagellar antigens. Of particular interest is Bacillus thuringiensis subspecies kurstaki and subspecies aizawai used for the control of lepidopteran pest insects, Bacillus thuringiensis subspecies israelensis used for the control of dipteran pest insects and Bacillus thuringiensis subspecies tenebrionis used for the control of coleopteran pest insects.
The first isolation of a coleopteran toxic Bacillus thuringiensis was reported in 1983 (A. Krieg et al., Z.and.Ent. 96, 500-508, European Patent Publication EP 0149162 A2).
The isolate, which was designated Bacillus thuringiensis subsp. tenebrionis, has been deposited with the German Collection of Microorganisms under accession number DSM 2803. Bacillus thuringiensis subsp. tenebrionis was isolated in 1982 from a dead pupa of the mealworm, Tenebrio molitor (Tenebrionidae, Coleoptera). The strain produces within each cell one spore and one or more insecticidal parasporal crystals which are of flat platelike form with an edge length of about 0.8 .mu.m to 1.5 .mu.m. It belongs to serotype H8a,8b and pathotype C of Bacillus thuringiensis (Krieg et al., System.Appl.Microbiol. 9, 138-141, 1987, U.S. Pat. No. 4,766,203, 1988).
It is only toxic against certain leaf eating beetle larvae (Chrysomelidae), but ineffective against caterpillars (Lepidoptera), mosquitoes (Diptera) or other insects.
Bacillus thuringiensis subsp. tenebrionis has been shown to be an effective control agent for the colorado potato beetle larvae. After uptake of crystals and spores from Bacillus thuringiensis subsp. tenebrionis or isolated crystals larvae, and to a certain extent adults, of the colorado potato beetle (Leotinotarsa decemlineata) stop feeding. Larval stages L1-L3 die within 1-3 days (Schnetter et al., in "Fundamental & applied aspects of invertebrate pathology", eds. R.A. Samson et al., Proceedings of the 4th Int. colloquium of Invertebrate Pathology, p. 555, 1986).
It has recently been shown that Bacillus thuringiensis subsp. tenebrionis in addition to the coleopteran active crystal also produces another parasporal crystal that is spindle-, speroidal or plateshaped (A.M. Huger & A. Krieg, J.Appl.Ent. 108, 490-497, 1989). The activity of the second crystal is not yet known.
Four commercial products of Bacillus thuringiensis subsp. tenebrionis have been developed for the control of coleopteran pests. NOVODOR.RTM. from Novo Nordisk A/S, TRIDENT.RTM. from Sandoz, and DiTerra.RTM. from Abbott Laboratories Inc., and Foil.RTM. from Ecogen.
The isolation of another coleopteran toxic Bacillus thuringiensis strain was reported in 1986 (Hernnstadt et al. Bio/Technology vol. 4, 305-308, 1986, U.S. Pat. No. 4,764,372, 1988). This strain, designated "Bacillus thuringiensis subsp. san diego", M7, has been deposited at the Northern Regional Research Laboratory, USA under accession number NRRL B-15939. A commercial product based on "Bacillus thuringiensis subsp. san diego" has been developed by Mycogen Corp.
Comparative studies of Bacillus thuringiensis subsp. tenebrionis, DSM 2803 and "Bacillus thuringiensis subsp. san diego", NRRL-B 15939 including phenotypic characterization of the vegetative cells, characterization of the toxic parasporal crystal and analysis of plasmid DNA have, however, shown that "Bacillus thuringiensis subsp. san diego" apparently is identical to the formerly isolated strain DSM 2803, Bacillus thuringiensis subsp. tenebrionis (Krieg et al.: J.Appl.Ent. 104, 417-424, 1987). Furthermore, the nucleotide sequences and deduced amino acid sequences of the coleopteran active delta endotoxin genes from Bacillus thuringiensis subsp. tenebrionis and "Bacillus thuringiensis subsp. san diego" are identical.
Under the same culture conditions the above-mentioned second type of crystals are also synthesized by "Bacillus thuringiensis subsp. san diego" (A.M. Huger & A. Krieg, J.Appl.Ent. 108, 490-497, 1989).
According to H. de Barjac & E. Frachon (Entomophaga 35(2), 233-240, 1990) the "san diego" isolate is similar to "tenebrionis" and it cannot be justified to regard it as a different subspecies.
The utility of Bacillus thuringiensis strains for the control of coleopteran pests is dependent upon efficient and economical production of the coleopteran active toxins and the potency of the product produced. This in turn is dependent upon the amount of delta endotoxins which can be produced by fermentation of the coleopteran active Bacillus thuringiensis strains.
B. thuringiensis has been used for many years for the production of insecticides, but although mutants of B. thuringiensis with increased delta-endotoxins yield would be advantageous, no such mutants have previously been described. Mutants producing higher yields of delta-endotoxins would give a more efficient and economical production of B. thuringiensis toxins and a possibility for manufacture of B. thuringiensis products with increased potency at equal cost. This in turn would be an advantage for the user as reduced volumes of pesticide formulation have to be stored and handled for a given acreage. In addition, the users will have less container material to dispose of, thereby reducing the impact on the environment.
Improvements of the production of delta endotoxin by Bacillus thuringiensis subsp. tenebrionis through mutation have not previously been reported.
One problem associated with the use of especially B. thuringiensis subspecies tenebrionis in controlling beetle larvae has been the relatively low potency or strength of the preparations requiring the application of relatively large amounts of preparation to the areas to be treated, such as 5 to 10 liter/ha compared to 1 to 2 liter/ha of most other B. thuringiensis products and most other insecticides.
Consequently a recognized need for products of improved strength exists.
One way to overcome this problem would be to concentrate the preparations. However, this would add considerably to the production cost in comparison to the savings obtained in storage and transportation.
A much more elegant solution would be to create mutants of existing B. thuringiensis strains capable of producing substantially larger amounts of delta endotoxins per cell.